Modified ent-Abietane Diterpenoids from the Leaves of Suregada zanzibariensis

The leaf extract of Suregada zanzibariensis gave two new modified ent-abietane diterpenoids, zanzibariolides A (1) and B (2), and two known triterpenoids, simiarenol (3) and β-amyrin (4). The structures of the isolated compounds were elucidated based on NMR and MS data analysis. Single-crystal X-ray diffraction was used to establish the absolute configurations of compounds 1 and 2. The crude leaf extract inhibited the infectivity of herpes simplex virus 2 (HSV-2, IC50 11.5 μg/mL) and showed toxicity on African green monkey kidney (GMK AH1) cells at CC50 52 μg/mL. The isolated compounds 1–3 showed no anti-HSV-2 activity and exhibited insignificant toxicity against GMK AH1 cells at ≥100 μM.

consistent with the molecular formula, C 20 H 24 O 4 , suggesting nine double-bond equivalents. The compound gave a UV absorption at λ max 264 nm, supporting the occurrence of an α,β-unsaturated carbonyl moiety, typical for diterpenoid lactones. 8,9 Its strong IR absorption bands at 3460 and 1705 cm −1 indicated the presence of hydroxy and carbonyl groups, respectively. The 1 H NMR spectrum (Table 1, Figure S1, Supporting Information) of 1 displayed a signal at δ H 6.35 (H-14) corresponding to a trisubstituted alkene, signals at δ H 5.  19), and 1.13 (H- 20)]. The corresponding carbons were identified using the HSQC spectrum ( Figure  S4, Supporting Information). The 13 C NMR spectrum (Table 1, Figure S2, Supporting Information) showed signals corresponding to 20 carbons, with chemical shifts compatible with a diterpenoid. 8,10,11 Two carbonyl groups resonated at δ C 210.9 (C-1) and 175.7 (C-16), which are typical for a ketone and a lactone, respectively.
The anti-HSV-2 activity and the cytotoxicity of the leaf crude extract and of compounds 1−3 are shown in Figures 4  and 5, respectively. The crude extract exhibited anti-HSV-2

Scheme 1. Plausible Biogenesis of Zanzibariolides A (1) and B (2)
Journal of Natural Products pubs.acs.org/jnp Article activity with an IC 50 of 11.5 μg/mL, while it reduced GMK AH1 cell viability by 50% (CC 50 ) at 52 μg/mL (Figure 4), giving a selectivity index CC 50 /IC 50 = 4.5. Hence, some components of the crude extract may possess anti-HSV-2 activity at noncytotoxic concentrations. Encouraged by these data, compounds 1−3, purified from this extract, were tested for their bioactivities. None exhibited anti-HSV-2 activity at a concentration up to 100 μM (Figure 5a). Compounds 1−3 were evaluated also for their ability to inhibit infection of A549 cells by the tick-borne encephalitis virus (TBEV) and infection of HeLa cells by the human rhinovirus type 2 (HRV-2) (page S23, Supporting Information). Under the concentration range tested (0.032−100 μM) compounds 1−3 exhibited no anti-TBEV nor HRV-2 activities. These compounds showed very little or no toxicity for GMK AH1 cells at ≥100 μM ( Figure  5b). Nonetheless, the potential cytotoxic effect of the leaf crude extract at 100 μg/mL raises safety concerns as the concoction of leaves from the plant is used in folk medicine for various ailments. 1,3 On the other hand, compound 3 has previously been reported to exhibit significant activity against α-glucosidase 22 and to be toxic (IC 50 1.78 μM) against human acute monocytic leukemia cells (THP-1). 23 Compound 4 was not tested for anti-HSV-2 activity, as it was isolated in low amount; however, it is known to exhibit significant antiinflammatory activity by inhibition of PGE2 and IL-6 secretion. 21 ■ EXPERIMENTAL SECTION General Experimental Procedures. Optical rotations were determined using a 341 LC OROT polarimeter at 589 nm and 24.0°C , whereas ECD spectra were acquired on a JASCO J-810, Rev.1.00, spectropolarimeter. UV spectra were obtained using CH 3 OH as the solvent on a Shimadzu UV-1650PC UV/vis spectrophotometer. Infrared (IR) spectra were recorded on a PerkinElmer Spectrum FT-IR spectrometer using liquid samples. NMR spectra were acquired either on an Agilent 400MR 400 MHz spectrometer equipped with a OneNMRProbe or on a Bruker Avance Neo 600 MHz spectrometer equipped with a TCI cryogenic probe and were processed using MestreNova (v14.0.0). Chemical shifts were referenced to the residual of carbon and proton signals of the deuterated solvents (CDCl 3 δ H 7.26 and δ C 77.16) as internal standard. Assignments were based on 1D ( 1 H and 13 C) and 2D (COSY, HSQC, HMBC, TOCSY, and NOESY) NMR spectra. Mass spectra were acquired on a Waters Micromass ZQ Multimode Ionization ESCI in ESI mode, connected to an Agilent 1100 series gradient pump system and a C18 Atlantis T3 column (3.0 × 50 mm, 5 μm), and using Milli-Q H 2 O−MeOH (5:95 to 95:5, with 1% HCO 2 H and a flow rate of 0.75 mL/min over 6 min). HRESIMS spectra were obtained with a Q-TOF-LC/MS spectrometer using a 2.1 × 30 mm 1.7 μM RPC 18     Extraction and Isolation. The air-dried leaves of S. zanzibariensis were ground to a fine powder to obtain a 1603 g sample, which was soaked three times in 3 L of CH 3 OH−CH 2 Cl 2 (7:3) at room temperature for 48 h, yielding a total of 74 g of crude extract after evaporation under reduced pressure at 40°C. The crude extract (71 g) was adsorbed onto silica gel (1:1) and loaded on a silica gel 60 (230−400 mesh) column. Gravitational elution was performed with a gradient of increasing polarity using EtOAc (0−100%) in isohexane, by collecting 92 fractions. Chromatographic separation at 30% EtOAc−isohexane gave fractions 26−34, which were combined and purified on a Sephadex LH-20 column (CH 3 OH−CH 2 Cl 2 , 1:1) to obtain a subfraction that was further separated with preparative TLC (silica gel) with EtOAc−isohexane (1:5) to afford β-amyrin 7 (4, 4 mg). Combined fractions 38−46 that were obtained at 40% from column chromatography were purified on a Sephadex LH-20 column (CH 3 OH−CH 2 Cl 2 , 2:3) and further washed with isohexane to give simiarenol 6 (3, 16 mg); 6 mg of this sample was crystallized from CH 2 Cl 2 −isohexane (1:1). Fractions 48−55 (obtained with 50% EtOAc−isohexane) were combined and washed with isohexane and further crystallized from CH 2 Cl 2 −isohexane (1:1) to afford zanzibariolide A (1, 236 mg) as white needle-like crystals. Furthermore, the combined fractions 62−84 eluted with 60−80% were washed with isohexane and crystallized from CH 2 Cl 2 −isohexane (1:1), affording zanzibariolide B (2, 1800 mg) as white needle-like crystals.
Zanzibariolide X-ray Crystal Structure Analysis. Single-crystal X-ray data for 1 and 2 were measured using a Rigaku SuperNova dual-source Oxford diffractometer equipped with an Atlas detector using mirrormonochromated Cu Kα (λ = 1.541 84 Å) radiation. The data collection and reduction were performed using the program CrysAlisPro, 24 and a numerical absorption correction based on Gaussian integration was applied The structure was solved with intrinsic phasing (ShelXT) 25 and refined by full-matrix least-squares on F 2 using the Olex2 software, 26 which utilizes the ShelXL module. 27 Anisotropic displacement parameters were assigned to non-H atoms. All C−H hydrogen atoms were refined using riding models with a U eq (H) of 1.5U eq (C) for methyl groups and a U eq (H) of 1.2U eq (C) for all other C−H groups. Single-crystal X-ray diffraction measurements for compound 3 were performed using graphite-monochromatized Mo Kα radiation (λ = 0.710 73) using a Bruker D8 APEX-II equipped with a CCD camera. Data reduction was performed with SAINT. Absorption corrections for the area detector were performed using SADABS. The structure was solved by direct methods and refined by full-matrix least-squares techniques against F 2 using all data (ShelXT, ShelXS). 27 All non-hydrogen atoms were refined with anisotropic displacement parameters. Hydrogen atoms were constrained in geometric positions to their parent atoms using OLEX2. 28 Diffuse contribution to diffraction in 3 was accounted for by using solvent masking. 29 The X-ray structures of 1 (CCDC 2181946), 2 (CCDC 2181947), and 3 (CCDC 2118304) have been deposited at the Cambridge Crystallographic Data Centre. Copies of the data can be obtained, free of charge, on application to Director, CCDC, 12 Union Road, Cambridge CB2 IEZ, UK (fax: + 44-(0)1223-336033 or email: deposit@ccdc.cam.ac.uk).
Crystal Antiviral Assay. African green monkey kidney epithelial cells 30 were employed for screening of antiviral and cytotoxic activities of both crude extracts and pure compounds isolated therefrom. The HSV-2 333 strain 31 was used. An HSV-2 plaque reduction assay was used to determine the effects of the plant extract and compounds on HSV-2 infectivity in GMK AH1 cells. 32 Briefly, the plant extract and all tested compounds were solubilized in DMSO, and the stocks (10 mg/mL) were stored at −20°C. Prior to the assay, the test samples were subjected to serial 5-fold dilutions in Eagle's minimum essential medium supplemented with 1% penicillin/streptomycin and 1% Lglutamine stocks (EMEM-M) to obtain a concentration range 1.6− 1000 μg/mL (extract) or 1.6−1000 μM (compounds). The control sample comprised various concentrations of DMSO solvent. The GMK AH1 cells were seeded in 24-well plates, and confluent, 3-dayold monolayers (ca. 3.7 × 10 5 cells/well) were used. The supernatant culture medium was removed, the cells were rinsed once with 200 μL of EMEM-M medium, and 400 μL of fresh EMEM-M was added. Then, the cells in duplicate wells received 50 μL of serial 5-fold dilutions of extract or compounds and after gentle shaking were left at 37°C in a humidified atmosphere comprising 5% CO 2 (the CO 2 incubator). Subsequently, 50 μL of EMEM-M medium comprising 100 plaque forming units of HSV-2 333 strain was added to each well, and following gentle shaking, the cells were left in the CO 2 incubator for 90 min. Then, the supernatant medium was removed, and the cells were overlaid with 750 μL of a 1% solution of methyl cellulose in EMEM-M (supplemented with 2% fetal calf serum) that contained the same concentrations of the test extract or compounds. Following incubation of cells for 3 days in the CO 2 incubator, the overlay medium was removed and the cells were stained with 1% crystal violet solution to visualize the viral plaques.
Cytotoxicity of the test extract or compounds for GMK AH1 cells was assayed as described by Said et al. 32 Briefly, 3-day-old monolayer cultures of GMK AH1 cells growing in 96-well cluster plates were used. The culture medium was removed, the cells were rinsed with 200 μL of EMEM-M medium, and 50 μL of fresh EMEM was added. Subsequently, 50 μL of EMEM-M comprising the test samples at 5fold dilutions was added in duplicate wells. The final concentrations of the extract and compounds were 100, 20, 4, 0.8, 0.16, and 0 (DMSO control) μg/mL (extract) or μM (compounds). Following incubation of cells with the test samples for 3 days in the CO 2 incubator, 15 μL of the CellTiter 96 AQueous One Solution reagent (Promega, Madison, WI, USA) was added. After shaking, the cells were left in the CO 2 incubator for a further 1 h, and absorbance at 490 nm was recorded. ■ ASSOCIATED CONTENT * sı Supporting Information